Isolation of Phenolic Derivatives and Essential Oil Analysis of Prangos ferulacea (L.) Lindl. Aerial Parts.

Prangos ferulacea (L.) Lindl. (Apiaceae) is a medicinal plant distributed in Mediterranean regions, Caucasia and southwest of Asia. In the present study phytochemical constituents of the extract obtained from the aerial parts of P. ferulacea were investigated using various chromatographic and spectroscopic methods. Essential oil of the plant aerial parts was also analyzed using GC-MS. Five phenolic derivatives, isoimperatorin (1), ferudenol (2), caffeic acid glucosyl ester (3), isorhamnetin-3-O-β-D-glucopyranoside (4) and quercetin-3-O-β-D-glucopyranoside (5) were isolated from the aerial parts of P. ferulacea and their structures were elucidated using 1H-NMR, 13C-NMR, EI-MS and UV spectral analyses. Twenty-seven compounds were also identified in the essential oil of plant aerial parts, of which β-pinene (43.1%), α-pinene (22.1%) and -δ3-carene (16.9%) were characterized as main compounds. The results of this study introduce P. ferulacea as a source of potentially bioactive phenolic compounds and suggest it as an appropriate candidate for further studies.


Introduction
The genus Prangos Lindl. from Apiaceae family, consists of about 30 species, mainly distributed in Irano-Turanian phytogeographic region (1). In flora of Iran, this genus is represented by 14 species including Prangos ferulacea (L.) Lindl. (2). P. ferulacea is a perennial plant with up to 1.5 m height which grows in Eastern Europe, Turkey, Caucasia and southwestern Asia (2). This species is known as «Jâshir» in Iran and its aerial parts are used traditionally as laxative and against ruminant parasites (3,4). In eastern Anatolia, the aromatic aerial parts of P. ferulacea are used as a flavor in cheese and its stem is also used as digestive, antidiabetic and antihypertensive agent (5, 6). Beside, P. ferulacea is used as animal fodder because of its valuable nutritive properties (7).
In the present study, we report essential oil constituents and isolation of the five phenolic derivatives, isoimperatorin (1), ferudenol (2), caffeic acid glucosyl ester (3), isorhamnetin-3-O-β-D-glucopyranoside (4) and quercetin-3-Oβ-D-glucopyranoside (5) from the aerial parts of P. ferulacea growing in Northwest of Iran. To the best of our knowledge, this is the first report on isolation and structure elucidation of these phenolic derivatives from the P. ferulacea aerial parts.

Experimental
General procedures 1 H-NMR and 13 C-NMR spectra were acquired in CDCl 3 and DMSO-d 6 on a Bruker Avance DRX 400 spectrometer. EI-MS spectra were obtained on a Hewlett-Packard model 5973 HP system. UV spectra were recorded in methanol (and after the addition of shift reagents) on a CECIL 7250 spectrophotometer.
Silica gel (230-400 mesh, Merck), fully endcapped RP-18 (230-400 mesh, Fluka) and Sephadex LH-20 (Fluka) were applied for column chromatographies. Preparative thin layer chromatography (PTLC) was also performed on handmade silica gel 60 GF 254 (Merck) plates. Pre-coated silica gel GF 254 aluminum sheets (Merck) were used for TLC and monitoring of the spots were carried out under UV (254 and 366 nm) and by spraying with anisaldehyde-H 2 SO 4 reagent followed by heating at 120°C for 5 min. All of the solvents were also obtained from Merck chemical company.

Plant material
The flowering aerial parts of P. ferulacea were collected from the Razi border, located in Khoy County (West-Azerbaijan Province, Northwest of Iran) in June 2014. The plant was identified by botanist Dr. Yousef Ajani, Institute of Medicinal Plants, Academic Center for Education, Culture and Research (ACECR), Karaj, Iran.

Extraction and fractionation
The air-dried aerial parts of P. ferulacea (1kg) were grinded and extracted exhaustively with MeOH by maceration method at the room temperature (10×12 L and 48 h each time). The combined extracts were concentrated by a rotary evaporator at 45°C. The obtained total extract (274.58 g) was fractionated successively with petroleum ether and chloroform using liquidliquid extraction, to get three main fractions, petroleum ether, chloroform and methanol (residue) fractions.

Isolation and purification of compounds
The petroleum ether fraction (12 g) was dissolved in petroleum ether (200 ml) and transferred into a flask. Following the addition of ethanol (30 ml) to above solution, a white precipitate was appeared which was then separated by filtration and named fraction E2.

Isolation of essential oil
Essential oil of the plant aerial parts was extracted using hydro-distillation for 4 h by a Clevenger-type apparatus. The prepared oil was subsequently dried over anhydrous sodium sulfate and stored at 4°C in the dark until analysis.

GC-MS analysis
An Agilent 6890 gas chromatograph (Column: BPX5, 30 m × 0.25 mm (id), 0.25 µm film thickness) equipped with a MS detector (Agilent 5973, EI mode at 70 eV, 220 °C) was applied for the essential oil analysis. The flow rate of carrier gas (Helium) was 0.5 ml min -1 . The oven temperature was raised from 50 °C to 240 °C at a rate of 3 °C per minute and then raised to 300 °C at a rate of 15 °C and finally maintained at 300 °C for 3 min. The injection temperature was 290 °C, and the oil sample (1.0 µL) was injected with a split ratio of 1:30. The Kovats retention indices (KI) of the compounds were calculated using a homologous series of n-alkanes (C 8 -C 30 ) injected in conditions equal to the samples. Identification of the chemical constituents was performed using Wiley7n.l online library, as well as by direct comparison of their MS spectra and KIs with data published in the literature for standard compounds (29).

Results
Phytochemical investigation of the aerial parts of P. ferulacea yielded the isolation of five compounds. The structures of isolated compounds were established as isoimperatorin (1), ferudenol (2), caffeic acid glucosyl ester A dash (-) indicate not reported. a Hydrodistillation, b Steam distillation.
Essential oil analysis of the P. ferulacea aerial parts resulted to the identification of twenty seven compounds, mainly β-pinene (43.1%), α-pinene (22.1%), δ-3-carene (16.9%) (Table 1). Osthole, a pernylated coumarin was identified in the plant essential oil with the relative percentage of 1.28%. This compound has been previously reported from the roots of P. ferulacea with antispasmodic effects on ileum contraction (11). Previous reports on essential oil composition of the different parts of P. ferulacea have been summarized in Table 2. Sefidkon et al. (1998) reported β-pinene (22.9%), δ-3-carene (16.0%) and α-pinene (12.6%) as the main compounds of the essential oil of P. ferulacea aerial parts from Isfahan (center of Iran) (16). The comparison of the results represented in this paper with the former study on P. ferulacea essential oil from Isfahan province (Iran) revealed that α-thujene, α-pinene, camphene, β-pinene, β-myrcene, δ-3carene, α-terpinene, p-cymene, (Z)-β-ocimene, (E)-β-ocimene, γ-terpinene, β-caryophyllene and α-bisabolol are the compounds present in both essential oil samples (16). Furthermore, in another study on essential oil of the plant flowering aerial parts from East-Azerbaijan (northwest of Iran), (E)-anethole (95.0%) and α-pinene (1.2%) were characterized as its main compounds (17). (E)-anethole, an aromatic major compound of the last mentioned study, however, was not identified in our analysed oil sample (17). The present study reports the monoterpene hydrocarbons (94.5%) as main group of constituents in P. ferulacea essential oil, whereas two previous reports from Isfahan and East-Azerbaijan provinces (Iran) reported the level of monoterpene hydrocarbons at 64.0 and 2.7%, respectively (16, 17). Genetic variations and climatic conditions could be considered as main factors involved in the chemical differences in composition of the essential oils obtained from P. ferulacea aerial parts (52).

Conclusion
The results of present study on isolation of bioactive phenolic derivatives (1-5) from the aerial parts of P. ferulacea emphasize the therapeutic potentials of this medicinal species and suggest it as an appropriate candidate for further biological and pharmacological research. This study also introduces P. ferulacea as a plant with monoterpene rich oil and report β-pinene (43.1%) as the main compound of its aerial parts oil.